Cloning And Function Verification Of The Antagonistic-related Gene Cluster Of Enterobacter Cloacea B8

Enterobacter cloacae B8 is a broad spectrum antagonistic bacterium isolated from rice leaf. It was found to have strong antagonistic activity, firstly using Xanthomonas oryzae pv. oryzae as indicator. Satisfactory results had been obtained on anti-rice bacterial leaf brilliant in net house and in field. Further researches showed that B8 was also antagonistic to not only other plant bacterial pathogens, but also human or animal bacterial pathogens such as MRSA. Therefore, E. cloacae B8 and its antagonistic substances would have potential application on both rice bacterial leaf brilliant and many other bacterial diseases of plants, animals and human. In this research, we focused on the molecule antagonistic mechanism of this E. cloacae B8 strain, which would offer theoretical supports for the better use of this strain for biological control.Two antagonistic mutant strains B8F, B8B of B8 were isolated with transposon mediated mutagenesis and two plasmids pTLF, pTLB were selected with tagging method. The 735bp sequence of the F fragment in pTLF was obtained after subcloning, and the 2608bp sequence of the B fragment in pTLB was obtained with GPS-1 system. To obtain sequence of unknown region, chromosome walking was adopted in our research. Different B8 genomic cassette libraries were constructed as templates and PCR were performed using cassette primer CP and specific primers designed according to known sequences of the F fragment and the B fragment. A 2586bp sequence left of the F fragment (or the Tn5 insertion site) and 644bp sequence right of that were obtained after 4 rounds of chromosome walking. A 3985bp F contig was assembled together with sequence of the F fragment. 2003bp sequence right of the B fragment was obtained after 2 rounds of chromosome walking, and a 4611bp B contig was assembled. Bio-informatics analysis found that the F contig encoded four ORFs and had high homology to admL, admM, admN and admO genes, respectively, of Pantoea agglomerans andrimid biosynthetic gene cluster (AY 192157). The gene knocked out by Tn5 insertion was named anrF, corresponding to admM gene (polyketide synthase, ORF 2583bp), and the insertion site was at 2368bp, 214bp before stop coden that is, of the anrFgene. Bio-informatics analysis also showed that B contig encoded 7 ORFs, 3 of thatIVwere at left of Tn5 insertion site. They were a 200bp gene fragment of GAPDH, 390bp ORF of probably transcriptional regulator and a 450bp ORF of unknown function. There was a 600bp fragment, with no apparent ORF between the first and the second ORF, that had some homology to the high pathogenicity island of Yersinia enterocolitica. Another 4 ORFs were at right. 3 of that had high homology to admA, admB and admC genes, respectively, of the andrimid biosynthetic gene cluster. A 408bp ORF, named anrP, was identified in a place 286bp upstream of 'admA' gene. The Tn5 insertion site was at non-coding region 894bp upstream of 'admA', or 200bp upstream of anrP gene. The anrF gene and the AnrB fragment knock-out by Tn5 insertion were verified with PCR amplification of the fragments and ends sequencing. It was concluded that the antagonistic related genes of E. cloacae B8 had been cloned.Long and accurate PCR was employed in our study to further verify the existance of similar antagonistic related cluster of the andrimid biosynthetic gene cluster in E. cloacae B8. A 12.5kb fragment between the 'admA' and the anrF genes was amplified using B2370-90 and F2000 primers and EX Tag. The PCR product was digested to three fragments of BA1, BA2 and BA3 with the restriction enzyme Hind III. The BA2 fragment with Hind III cohesive termini at both ends was cloned to pUCIS vector that digested with Hind III and dephosphorylated with CIAP and sequenced. Based on the entire sequence of BA2 fragment, primers were designed and BAT and BA3' fragments were amplified. The BAT fragment was then also cloned and sequenced. A 4997bp BA contig was assembled from both sequences of BAT and BA2. Bio-informatics analysis showed that the BA contig had highly homology to se...